title: Purification and characterization of Haemophilus influenzae D-lactate dehydrogenase.
abstract: Haemophilus influenzae D(-)-lactate dehydrogenase (D(-)-lactate:NAD oxidoreductase; EC 1.1.1.28) was purified to electrophoretic homogeneity using salt fractionation, hydrophobic and dye affinity chromatography. The enzyme was purified 2100-fold with a 14% recovery and a final specific activity of 300 units/mg protein. The enzyme was demonstrated to be a tetramer of Mr 135,000. The enzyme catalyzed the reduction of pyruvate to give exclusively D(-)-lactate using NADH as coenzyme. The reaction catalyzed was essentially unidirectional, with the oxidation of D-lactate in the presence of NAD proceeding at less than 0.2% the rate of pyruvate reduction. Kinetic parameters for the reduction of pyruvate were determined for NADH and four structural analogs of the coenzyme. Coenzyme-competitive inhibition by adenosine derivatives indicated the presence of regions in the coenzyme binding site interacting with the adenosine and pyrophosphate moieties of the coenzyme. The purified enzyme was sensitive to oxidation and was effectively inactivated by sulfhydryl reagents. Conversion of D-lactate to pyruvate catalyzed by a membrane-bound D-lactate oxidase was demonstrated in cell-free extracts of H. influenzae.
authors.affiliation: Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg 24061-0308.
authors.forename: A
authors.initials: A
authors.lastname: Denicola-Seoane
authors.forename: B M
authors.initials: BM
authors.lastname: Anderson
mesh_terms: D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006193:Haemophilus influenzae / Q000201:enzymology*; D007536:Isomerism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D011489:Protein Denaturation; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D009243:NAD; D007770:L-Lactate Dehydrogenase